Introduction

Methods

Dissections

Fixing

Antibodies

Mounting

Microscopy

Fluorescence and brightfield microscopy
After mounting, we waited an appropriate amount of time for the slides to dry. All slides were initially looked at under a Zeiss Axiovert 200 fluorescence microscope. We viewed a total of 59 stained slides under the fluorescent microscope and chose selected slides to be imaged in more detail using an Olympus Fluoview 1000 confocal microscope.

Confocal microscopy
After locating the area of interest on each slide using regular fluorescence and brightfield microscopy we used the confocal imaging software. To optimize the image we adjusted the focus, offset, voltage, and gain. To generate the images below we used a Kalman filter to minimize background noise and a longer exposure time to obtain high quality images. We additionally took Z stacks of the slides in order to view a 3D perspective of the tissue.

Images

C. borealis eye (near retina) and pericardial organ labeled with DH

Results

2009 Class Group

Jan Plan 2009 - Advanced Neurobiology BI474

Course Goals:

To learn techniques such as microdissection, cell culture, brightfield microscopy, fluorescence microscopy, confocal microscopy, and image analysis.
To apply these techniques to a collaborative group project.
To gain expertise in an area of neuroscience research; to design and conduct a research project; to analyze, interpret, and present results.
To construct and update this website as an informative and instructive tool for the course.

Crustacean X-organ cell with neurites, 400x, Phase	Lola the Microscope, Funded by INBRE-Maine

Mount Desert Island Biological Laboratory: We spent 2 days there working with their confocal microscope in an INBRE-funded mini-session.	Jackson Laboratory: We also toured the impressive imaging facility at Jackson with our host Jim Denegre

More MDIBL Pix:


[Above] MDIBL Coop [Below] Confocal image of crustacean eyestalk X-organ	[Above] Christina, Jen, Ruthie, Kimberly, Ben, Sharonda, Vic, Andrea, and Angela Parton - our fearless confocal wizard (Taking the pic: Amy)